The purpose of the proposed work is to examine the role played by the contractile protein tropomyosin in stress fiber formation in cultured mammalian cells. In our previous work we have shown that normal rat kidney cells, which spread over the substrate and adhere tightly to it, contain extensive basal networks of actin filament bundles termed "stress fibers". WE can demonstrate biochemically and immunologically that tropomyosin is associated with the actin filaments in these bundles. A virally transformed NRK cell, termed 442, does not spread on the substrate and adheres poorly to it. These cells do not assemble stress fibers, and they appear to lack tropomyosin completely. Since it is known from the work of others that tropomyosin can stabilize actin filaments and is required for the organization of actin filaments in developing muscle, we hypothesize that a virally induced lack of tropomyosin in 442 cells is responsible for the absence of stress fibers in the cell. We will test this hypothesis by using direct microinjection and red cell fusion methods to re-introduce tropomyosin into 442 cells. The injected protein will be traced by a number of methods, including conjugation to fluorochromes. We will monitor injected cells in order to determine if they acquire the ability to assemble stress fibers and to spread over the substrate in a more normal fashion. We will use the same injection procedures to introduce anti-tropomyosin antibodies into NRK cells in order to determine if inactivation of the cell's normal complement of tropomhosin causes disassembly of stress fibers and an alteration in cell shape. Using these paired cells, for which many characteristics of the cytoskeleton are already defined, we believe that we are in a unique position to analyze the mechanism of stress fiber assembly and the role it plays in cell adhesion and maintenance of cell shape. In the longer term, we hope to arrive at a better understanding of the mechanism of viral transformation and how to deal with it.